Neutrophil serine proteases are a family of effector molecules of the innate immune system that are important for protecting against invading pathogens. Members of this trypsin-fold protease family, neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), not only play critical roles in neutrophil-mediated clearance of invading microbes (Reeves et al., Nature, 2002, 416:291-297; Weinrauch et al., Nature, 2002, 417:91-94; Belaaouaj et al., Nat Med, 1998, 4:615-618) and inflammation (Pham et al., Nat Rev Immunol, 2006, 6:541-550) but can also participate in the pathogenesis of various diseases (Magrone et al., Curr Pharm Des., 2012, 18(12):1609-19 and Pham et al., Int J Biochem Cell Biol., 2008, 40(6-7):1317-1333). Recently, the serine protease PRSS57, originally discovered by yeast signal trap screening and computational mining of human cDNA libraries (Clark et al., Genome Res., 2003, 13:2265-2270), was identified as the fourth NSP member and subsequently referred to as neutrophil serine protease 4 (NSP4) (Perera et al., Proc Natl Acad Sci USA, 2012, 109:6229-6234; Perera et al., J Immunol., 2013). Remarkably, NSP4 is highly conserved from bony fishes to human and predates the emergence of other NSPs, indicating that NSP4 likely plays fundamental roles in neutrophil biology (Perera et al., Proc Natl Acad Sci USA, 2012, 109:6229-6234; Perera et al., Expert Rev Clin Immunol, 2012, 8:501-503).
The relatively broad substrate specificities of NE, CG, and PR3 are well understood based on the detailed knowledge of their active site structures (Navia et al., Proc Natl Acad Sci USA, 1989, 86:7-11; Hof et al., EMBO J, 1996, 15:5481-5491; Fujinaga et al., J Mol Biol, 1996, 261:267-278.). However, NSP4 poses a conundrum in that, like trypsin, it cleaves substrates after arginine residues (Perera et al., Proc Natl Acad Sci USA, 2012, 109:6229-6234; Perera et al., J Immunol., 2013), but paradoxically has a primary sequence that predicts a very different elastase-like active site with preference for small aliphatic amino acids and is seemingly incompatible with the long P1-arginine side chain. Due to NSP4's long evolutionary lineage, and its distinctive active site, it is possible that NSP4 is an important protease for neutrophil function and may contribute to neutrophil-mediated disease or disorders. However, relative to other members of the neutrophil serine protease family, the role of NSP4 in neutrophil-mediated diseases, such as arthritis, is unknown. For instance, the combined deficiencies of NE and CG was required to confer full protection in the mouse collagen antibody-induced arthritis model (Adkison et al., J Clin Invest, 2002, 109:363-371). NE and CG are also each capable of processing and activating IL-33 (Lefrancais et al., Proc Natl Acad Sci USA, 2012, 109:1673-1678), which is a pro-inflammatory cytokine that promotes inflammatory arthritis (Xu et al., Proc Natl Acad Sci USA, 2008, 105:10913-10918). Similarly, the combined ablation of NE and PR3 was required to prevent the inactivation of progranulin (Kessenbrock et al., J Clin Invest, 2008, 118:2438-2447), an anti-inflammatory cytokine that alleviates inflammatory arthritis (Tang et al., Science, 2011, 332:478-484).
Resolving the paradox between the predicted elastase-like active site with the actual trypsin-like active site that is exhibited by NSP4 has the potential to provide structural features of the enzyme active site that could facilitate the development of specific NSP4 inhibitors. These NSP4 inhibitors may serve a need for treatment of neutrophil-mediated diseases or disorders where the underlying pathology is completely or partially due to the activity of NSP4.
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